Paracrine signals from mesenchymal feeder cells and regulating expansion and differentiation of hepatic progenitors using same

Abstract

A method is provided for controlling the survival, proliferation, and/or differentiation of hepatic progenitors in vitro by using specific types of mesenchymal feeder cells or one of more of the paracrine signals produced by those feeders.

1 . A method of propagating hepatic stem cells in vitro without inducing their differentiation comprising: culturing a population of isolated hepatic stem cells in serum-free culture media and on a layer of matrix components selected from the group consisting of hyaluronans, other unsulfated or poorly sulfated glycosaminoglycans (GAGs), unsulfated or poorly sulfated proteoglycans, embryonic collagens and embryonic basal adhesion molecules, and combinations thereof,
 in which the layer is essentially free of mature collagens, and in which the culturing propagates the hepatic stem cells without inducing their differentiation.   
   
   
       2 . The method of  claim 1  in which any or all of the matrix components are supplied by angioblast feeder cells, quiescent hepatic stellate feeder cells, HUVEC feeder cells, or a combination thereof. 
   
   
       3 . The method of  claim 1  in which the embryonic collagens are of type III. 
   
   
       4 . The method of  claim 1  in which the mature collagen is collagen type I. 
   
   
       5 . The method of  claim 1  in which the basal adhesion molecules comprise isoforms of laminin found predominantly in fetal tissues. 
   
   
       6 . The method of  claim 1  in which the GAGs other than hyaluronans are forms of chondroitin sulfates. 
   
   
       7 . The method of  claim 1  in which the proteoglycans are forms of chondroitin sulfate proteoglycans (CS-PGs). 
   
   
       8 . The method of  claim 1  in which the hepatic stem cells are obtained from fetal, neonatal, pediatric or adult liver. 
   
   
       9 . The method of  claim 8  in which the liver is human liver. 
   
   
       10 . The method of  claim 5  in which the laminin is at a concentration between about 0.1 to about 2 μg/cm 2 . 
   
   
       11 . The method of  claim 10  in which the laminin is at a concentration of about 1 μg/cm 2 . 
   
   
       12 . The method of  claim 3  in which the type III or IV collagens are individually at a concentration between about 0.1 to about 15 μg/cm 2 . 
   
   
       13 . The method of  claim 1  in which the layer comprises hyaluronans. 
   
   
       14 . A method of differentiating hepatic stem cells in vitro to hepatoblasts comprising: culturing a population of isolated hepatic stem cells in serum-free culture media and on a layer of matrix components selected from the group consisting of embryonic collagens, basal adhesion molecules, CS-PGs, and combinations thereof,
 in which the layer is essentially free of mature collagens, and in which the culturing propagates the hepatic stem cells without inducing their differentiation.   
   
   
       15 . The method of  claim 14  in which any or all of the matrix components are supplied by activated endothilia, activated hepatic stellate feeder cells, or both. 
   
   
       16 . The method of  claim 14  in which the embryonic collagen is type IV collagen. 
   
   
       17 . The method of  claim 14  in which the basal adhesion molecules comprise fetal isoforms of laminin. 
   
   
       18 . The method of  claim 14  in which the layer further comprises hyaluronans. 
   
   
       19 . The method of  claim 14  in which the hepatic stem cells are obtained from fetal, neonatal, pediatric or adult liver. 
   
   
       20 . The method of  claim 19  in which the liver is human liver. 
   
   
       21 . A method of differentiating hepatic stem cells or hepatoblasts in vitro into committed hepatocyte or biliary progenitors and progeny thereof comprising: culturing a population of isolated hepatic stem cells in serum-free culture media and on a layer of matrix components selected from the group consisting of sulfated proteoglycans, mature collagens, fibronectin, and combinations thereof,
 and in which the culturing induces the differentiation of the hepatic stem cells or hepatoblasts into committed hepatic or biliary progenitors and progeny thereof.   
   
   
       22 . The method of  claim 21  in which any or all of the matrix components are supplied by stromal feeder cells, activated hepatic stellate feeder cells, myofibroblast feeder cells, or combinations thereof. 
   
   
       23 . The method of  claim 21  in which the mature collagen is collagen type I. 
   
   
       24 . The method of  claim 21  in which the layer is substantially free of hyaluronans. 
   
   
       25 . The method of  claim 21  in which the hepatic stem cells are obtained from fetal, neonatal, pediatric or adult liver. 
   
   
       26 . The method of  claim 25  in which the liver is human liver. 
   
   
       27 . The method of  claim 21  in which the sulfated proteoglycans are heparan sulfate-PG or heparin-PG, or both. 
   
   
       27 . A container for propagation of hepatic progenitors comprising:
 (a) a container, and   (b) a layer of matrix components selected from the group consisting of hyaluronans, other unsulfated or poorly sulfated glycosaminoglycans (GAGs), unsulfated or poorly sulfated proteoglycans, embryonic collagens and embryonic basal adhesion molecules, and combinations thereof;   wherein the layer is essentially free of mature collagens; and   wherein the layer of matrix components substantially coats at least one surface of the container.   
   
   
       29 . The container of  claim 1  in which the container is a tissue culture plate, a bioreactor, a lab cell or a lab chip. 
   
   
       30 . A container for propagation of hepatic progenitors comprising:
 (a) a container, and   (b) a layer of matrix components selected from the group consisting of embryonic collagens, basal adhesion molecules, CS-PGs, and combinations thereof,   wherein the layer is essentially free of mature collagens; and   wherein the layer of matrix components substantially coats at least one surface of the container.   
   
   
       31 . A container for propagation of hepatic progenitors comprising:
 (a) a container, and   (b) a layer of matrix components selected from the group consisting of sulfated proteoglycans, mature collagens, fibronectin, and combinations thereof,   wherein the layer of matrix components substantially coats at least one surface of the container.